PICT offers a global approach for the discovery of enzymes and molecules of therapeutic interest and the understanding of their interactions with their target, which is important for the development of selective and effective drugs. This global approach is based on three complementary fields of expertise:
- The development of chemical libraries for generating molecular diversity and the optimization of ligands.
- Structure-based ligand design and optimization using bioinformatics, biophysics and structural biology.
These key competencies are applied to an interdisciplinary process allowing identification, design, synthesis and optimization of ligands that will target macromolecules of biological interest. PICT combines the know-how and the expertise of the existing facilities described below.
Structural biology plays a key role in structure-based ligand design and in the Fragment Based Ligand Discovery approach. PICT can provide the 3D structure determination of macromolecules both by X-ray crystallography and NMR approaches.
The macromolecular crystallography facility
X-ray crystallography is the primary method for determining three-dimensional structures of biological macromolecules and of their complexes with ligands at high resolution. The goal of the facility is to provide investigators with state-of-the-art instrumentation and expertise for:
- Protein characterization in solution
- Data Collection
- Structure Determination and Refinement
The macromolecular crystallography facility is staffed to carry out structural analysis on a service basis or to train and assist interested users in crystallization. Two dedicated engineers run the platform. In addition, projects may benefit from the expertise of the IPBS structural biophysics group ( link to research team). Staff can provide advice about how to obtain diffraction-quality crystal and evaluate initial crystals for their quality and the feasibility of structure determination.
The equipment operated within the facility includes nano-dispensing crystallization robots, automated imaging system, X-ray diffractometers and cryogenic systems. We also have regular access to French and European synchrotrons.
The NMR facility offers a complete set of NMR approaches aiming at characterizing structures, dynamics and interactions of biological molecules, in particular:
- Structural determination of biomolecules: from the chemical structure of biologically relevant medium size molecules to 3D structure determination of macromolecules and macromolecular complexes.
- Drug or ligand discovery: NMR based screening (STD, waterLOGSY, trNOEs etc..) and interaction site characterization (chemical shift mapping).
- Monitoring enzymatic reactions by NMR : determination of binding epitopes and measuring enzyme kinetics.
These studies may be carried out using high-resolution liquid-state NMR (1D-4D) on three Bruker spectrometers: 500 MHz, 600 MHz cryoprobe and 700 MHz, and a broad range of multinuclear probes. Solid-state NMR capacities on Bruker 500 MHz and 700 MHz spectrometers are also available, including a broad range of static and Magic Angle Spinning probes (12 KHz to 67 KHz spinning frequencies) double or triple resonances. Access to higher fields (800 to 1000 MHz) is possible via regional, national and European NMR facilities.
Two dedicated research engineers run the platform. They are in charge of the spectrometer maintenance according to ISO 9001 quality procedures. Upon request they will analyze the analytical problem proposed by the customers and propose the most suitable strategies and experiments. The access to the facility may be performed entirely by the engineers or in part by the customers after proper training.
In the case of complex analytical problems the expertise from the IPBS NMR groups (~15 researchers, link to research team) may be involved via research contracts.
The biophysical facility provides instrumentation and expertise for the characterization of macromolecules and molecular interactions using several state-of-the-art biophysical methods, including dynamic and static light scattering (DLS, SLS), circular dichroism (CD), differential scanning fluorimetry (DSF), and isothermal titration calorimetry (ITC).
- Oligomerization and molar mass determination (DLS and SEC-MALS-QELS)
- Assessment of protein folding, secondary structures (CD)
- Fluorescent monitoring of molecular interactions (DSF)
- Affinities and thermodynamic bases of molecular interactions (ITC)
The aim of the chemical library is to design and provide chemical libraries for screening step and to optimize the selected ligands through chemical synthesis.
The chemical facility is fully equipped with modern equipment to perform synthesis as well as analysis, physical characterization and purification of bioactive molecules. It possesses microwaves, hydrogenation and ozonolysis apparatus, automatic systems for slow addition of reactants, a multi-solvent purification system, several spectrometers (UV/VIS, fluorescence, IR) photochemical reactors, and oligonucleotide synthesizer and a bench mass spectrometer (MALDI-TOF). Thanks to its partnership with the "Institut des Technologies Avancées en Sciences du Vivant" (ITAV), the platform has access to automatic parallel synthesis work stations to operate classic or solid-supported chemistry: SLT 100 Chemspeed (up to 48 reactions in parallel) and SWave Chemspeed (up to 184 reactions).
The platform has also an operational HPLC platform open to academic or private customers. Specific material dedicated to this service is analytical (simple and UHPLC), semi-preparative (chiral or achiral) and preparative chains with multitype detectors allowing to work in normal or reverse silicagel phase or in supercritical flow. An additional autopurification HPLC chain coupled with a simple quadripole mass spectra is also available.
In addition, the chemical facility has access, through the physical chemistry platform of ICT (http://ict.ups-tlse.fr), to all the modern analytical equipment and techniques required for the authentication and characterization of the compounds that are produced (intermediate steps and final compounds): NMR, IR, mass spectrometry, microanalysis.
Peptide synthesis provides biologically active peptides used as functional tools for scientific research in areas such as immunology and biochemistry. The peptide synthesis service provides:
- Standard peptides synthesis from 5 to 45 amino acids.
- Original peptides synthesis: long chain peptides, amyloid peptides with or without mutations, hydrophobic sequences (transmembrane peptide sequences), labeled peptides (2D, 13C, 15N), cyclic peptides (disulfide or amide bridges), fluorescent peptides, peptides with unsatured amino acids for tritium labeling, glycosylated peptides, phosphorylated peptides, N-terminal and C-terminal modifications.
- Identification of potentially antigenic sequences in a protein to select peptides for antibody production (synthesis of peptides and antigen preparation).
Depending on the expected objectives, a feasibility study will be performed leading to a proposal and quotation. The strategy of the synthesis, carried out chemically on solid support, will be adapted to the requested sequence. The quality of the molecules synthesized will be checked by HPLC and mass spectrometry.
The bio/chemoinformatics facility provides computational resources aimed at solving the problem of searching for promising ligands of a specified target. This is mainly accomplished by performing (i) molecular dynamics simulations and (ii) virtual screening. The first approach enables the assessment of the global conformational stability of a complex and allows locating persistent contact points such as hydrogen bonds between the interacting molecules. The second approach allows identifying promising leads in the search for optimal ligands.
Two clusters and several GPU servers are available for computational tasks. They work independently, but are interconnected via Gigabit Ethernet network, which facilitates data transfers. Software resources are continuously updated.
The enzyme screening facility (ICEO) provides the expertise and the robotic resources necessary for the discovery of original enzymes, coming either from combinatorial engineering projects - a method used to modify or improve protein properties in order to adapt catalysts to special conditions of use – or after screening of microbial collections or genomic /metagenomic libraries.
To explore the diversity offered by such libraries (which can contain from 105 to 1013 clones), the development of robotic screening strategies, fast and reliable, are required. This is why ICEO is equipped with up-to-date high-throughput instrumentation and benefit from an adequate expertise in the domains of molecular diversity.
Creation of enzyme diversity
- genomic or metagenomic libraries
- directed evolution
- in silico protein design
- rational and/or semi-rational protein engineering
Enzymatic activity screening
- selection test setting
- enzymatic assay miniaturization for HTS
- liquid medium screening
- solid medium screening
- ad hoc substrate synthesis
- complete biochemical characterization of selected enzymes.
Thus, ICEO can offer very practical and technological outcomes but also can contribute to answer to critical scientific questions.